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1 Department of Microbiology & Cell Biology, Indian Institute of Science, Bangalore 560012, India
2 Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India
Correspondence
C. Durga Rao
cdr{at}mcbl.iisc.ernet.in
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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N72) was recently obtained from strains Hg18 and SA11 exhibiting approximately 17–20-, 150–200- and 13166–15800-fold lower DD50 (50% diarrhoea-inducing dose) values in suckling mice compared with that reported for the partially pure, full-length protein, a C-terminal M175I mutant and a synthetic peptide comprising aa 114–135, respectively, suggesting the requirement for a unique conformation for optimal functions of the purified protein. The stretch of approximately 40 aa from the C terminus of the cytoplasmic tail of the endoplasmic reticulum-anchored NSP4 is highly flexible and exhibits high sequence variation compared with the other regions, the significance of which in diarrhoea induction remain unresolved. Here, it was shown that every amino acid substitution or deletion in the flexible C terminus resulted in altered conformation, multimerization, trypsin resistance and thioflavin T (ThT) binding, and affected DLP binding and the diarrhoea-inducing ability of the highly diarrhoeagenic SA11 and Hg18 N72 in suckling mice. These studies further revealed that high ThT fluorescence correlated with efficient diarrhoea induction, suggesting the importance of an optimal ThT-recognizable conformation in diarrhoea induction by purified NSP4. These results based on biological properties provide a possible conformational basis for understanding the influence of primary sequence variations on diarrhoea induction in newborn mice by purified NSP4s that cannot be explained by extensive sequence analyses. The oligonucleotides used in this study are presented as a supplementary table available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and account for approximately 600 000 infant deaths annually (Parashar et al., 2006). The rotavirus genome consists of 11 segments of dsRNA, encoding six structural and six non-structural proteins. The virion is a triple-layered particle, with VP4 and VP7 constituting the outer layer. Virus particles devoid of the outer layer are referred to as double-layered particles (DLPs), with VP2 and VP6 forming the inner and intermediate layers, respectively (Estes, 2001).
NSP4, encoded by genome segment 10, is a multifunctional protein (Fig. 1a) and is essential for virus morphogenesis and pathogenesis (Lopez et al., 2005; Silvestri et al., 2005). With the N terminus of the 175 aa protein anchored in the endoplasmic reticulum (ER), the C terminus attains a cytoplasmic orientation (Estes, 2001). The N terminus contains an uncleaved signal sequence and three hydrophobic domains, H1, H2 and H3 (Fig. 1a). Whilst H1 (aa 7–21) is oriented in the lumen of the ER, H2 (aa 28–47) traverses the ER bilayer and H3 (aa 67–85) is embedded in the cytoplasmic side of the ER membrane with aa 45–175 constituting the extended cytoplasmic tail (CT), which exhibits all of the known important biological functions (Bergmann et al., 1989; Chan et al., 1988). Whilst the region spanning H3 to aa 137 exists as a tetrameric coiled-coil domain (CCD), the C-terminal region appears to be highly flexible (Deepa et al., 2007; Jagannath et al., 2006; Taylor et al., 1996).
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) in the context of the full-length protein or in fusion with glutathione S-transferase (GST) appears to be sufficient for DLP binding (Au et al., 1993; O'Brien et al., 2000; Taylor et al., 1993).
We have recently shown that cooperation between the N-terminal amphipathic helix located between aa 73 and 85 (AAH73–85) and the extreme C terminus in N72, a mutant NSP4 lacking the N-terminal 72 aa, from simian strain SA11 or bovine strain Hg18 promotes ordered multimerization (Jagannath et al., 2006). Mutations in the AAH73–85 or interspecies variable domain (ISVD) from aa 135 to 146 (Mohan & Atreya, 2000), deletions in the N terminus (N85 orN94) or substitution of the extreme C-terminal methionine in the context of N72 resulted in altered conformation, multimerization, resistance to trypsin and thioflavin T (ThT) binding, and severely affected DLP binding and diarrhoea-inducing properties with a 10–2000-fold increase in the 50% diarrhoea-inducing dose (DD50) compared with the highly diarrhoeagenic N72. These results suggested that the properties of NSP4 are dependent on a unique structurally and functionally overlapping conformational domain formed by cooperation between the N- and C-terminal regions of the CT (Jagannath et al., 2006).
NSP4 from strain SA11 and a synthetic peptide corresponding to aa 114–135 have been reported to induce dose-dependent diarrhoea in 6–7-day-old newborn mouse pups when injected intraperitoneally or intraileally (Ball et al., 1996). However, the synthetic peptide exhibited an approximately 800-fold higher DD50 compared with the full-length SA11 NSP4. Furthermore, a peptide comprising aa 112–175, secreted from rotavirus-infected cells, was reported to cause diarrhoea in suckling mice similar to the full-length protein (Zhang et al., 2000), suggesting that the N-terminal boundary of the enterotoxin activity could lie around residue 112. However, identification of the C-terminal boundary of the diarrhoea-inducing region and the precise role of the flexible C terminus in the biological functions of NSP4 remain unresolved. It has been presumed that the C-terminal boundary lies around aa 135 or 138, as the synthetic peptide of aa 114–135 induced diarrhoea (Ball et al., 1996) and mutations at positions 135 and 138 in the ISVD resulted in loss of virus virulence and diarrhoea-inducing ability of the protein (Zhang et al., 1998). Furthermore, the region C-terminal to the CCD exhibits a high degree of sequence variation compared with the other regions of the protein without correlation to virus virulence, and is highly flexible and susceptible to proteases due to its extended conformation (Jagannath et al., 2000, 2006; O'Brien et al., 2000). Hence, no role in diarrhoea induction has been considered. Although, the extreme C terminus is critical for DLP binding, iodination of tyrosines in microsome-localized NSP4 from virus-infected cells resulted in loss of DLP binding, suggesting the presence of a second site of interaction away from the extreme C terminus (Au et al., 1989).
To examine the role of the highly flexible C-terminal region from aa 139 to 175 in diarrhoea induction and DLP binding, and to identify the C-terminal boundary of the diarrhoea-inducing region and the presumed second site of DLP interaction, a large number of deletion and amino acid substitution mutants of the highly diarrhoeagenic SA11 and Hg18 N72, the longest form of NSP4 that could be expressed and purified in soluble form from Escherichia coli, were generated and their DD50 in suckling mice and DLP-binding properties were evaluated. The studies revealed that the flexible C terminus is a major determinant of the conformation and the biological properties of the CT of NSP4.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) rotavirus strains were grown for 3 days in the presence of 0.1 µg trypsin ml–1 in medium without serum. The virus in the supernatant from freeze–thawed infected cells was used for DLP preparation. Enzymes and reagents were from Roche Applied Science, Promega Biotech and GE Healthcare.
Cloning of NSP4 gene fragments in the expression vector pET22-NH.
Cloning of the NSP4 gene, deletion mutants N72 and N94, C-terminal mutants Hg18 N72C5 (Hgm6) and M175I (Hgm15) and ISVD mutants Y131S/T139S (dirm2) and T139S (dirm4) of Hg18 or SA11 and generation of the pET22-NH vector for expression of proteins in fusion with an N-terminal His tag have been described previously (Jagannath et al., 2006). Fragments corresponding to different deletions or amino acid substitutions were amplified by PCR using the wild-type (WT) N72 as template. The inserts were cloned in pET22-NH between the NdeI and HindIII sites. The nucleotide sequence was determined using T7 and M13 forward and reverse primers (Macrogen). Mutants and the nature of each mutation are described in Table 1.
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Expression, purification and analysis of the C-terminal mutant NSP4 proteins.
All of the mutant NSP4 proteins expressed in E. coli BL21(DE3) were highly soluble and were purified by Ni2+–nitrilotriacetic acid–agarose (Qiagen) affinity chromatography (Jagannath et al., 2006). The purity of the proteins was monitored by Tricine SDS-PAGE (Fig. 1b) or SDS-PAGE (Fig. 1c) and mass spectrometry. The approximate molecular masses of the native proteins were determined by size exclusion chromatography (SEC) using a Sephacryl S-200 column (GE Healthcare) on a Bio-Rad Biological DuoFlow system (Jagannath et al., 2006).
Mass spectrometry.
The molecular masses of the purified proteins and the trypsin-digested products were determined in an Ultraflex time-of-flight mass spectrometer (Bruker Daltonics) (Jagannath et al., 2006; Karas & Hillenkamp, 1988).
Circular dichroism (CD) spectroscopy.
For far-UV CD spectroscopy, proteins were dialysed against 5 mM Na3PO4 buffer (pH 7.4) containing 5 mM NaCl. CD spectra were recorded on a JASCO J-715 spectropolarimeter at a protein concentration of 10 µM. The molar residue ellipticity was calculated as described previously (Jagannath et al., 2006). Protein concentration was determined using A280 in 8 M urea and the extinction coefficient of each mutant (Jagannath et al., 2006). The percentages of 
-helical, β-sheet and random conformation contents were determined using the k2d program (Andrade et al., 1993). CD spectra were recorded at least three times for each mutant.
Thioflavin T (ThT) fluorescence assay.
A ThT fluorescence assay was performed three times for each protein at a final protein and ThT concentration of 10 µM. The excitation wavelength was 440 nm and emission was monitored from 450 to 600 nm (Ferrari et al., 2001; Jagannath et al., 2006; Naiki et al., 1989) in a Shimadzu RF-5301 PC spectrofluorometer at 25 °C.
NSP4 enterotoxin assay.
The DD50 of different NSP4 mutants was assessed in 5–7-day-old BALB/c mouse pups by injecting 1 pmol to 20 nmol of the protein intraperitoneally in 50 µl PBS. The DD50 and mean diarrhoeal scores were determined as described previously (Ball et al., 1996; Jagannath et al., 2006). For each dose, eight newborn mouse pups were used and the experiment was repeated three times with prior approval of the institutional ethics committee. Diarrhoea was noted between 30 min and 4 h after injection and was scored on a scale of 1–4 based on whether the stool was loose and yellow, semi-solid with mucus, watery or completely liquid, respectively (Ball et al., 1996). The fold increase in DD50 for different mutants of SA11 or Hg18 was calculated with reference to the DD50 of the N72 mutant of the respective strain that exhibited the lowest value.
Trypsin digestion of NSP4 proteins.
Proteins were incubated with trypsin at 37 °C, the trypsin-cleaved products were analysed by Tricine SDS-PAGE (Schagger & von Jagaw, 1987) and the molecular masses of the cleaved products were determined by mass spectrometry (Jagannath et al., 2006).
DLP-binding assay.
DLPs were prepared by CsCl density gradient equilibrium ultracentrifugation as described previously (Au et al., 1989). The purity of the DLPs was assessed by SDS-PAGE. The wells of a microtitre plate were coated with purified protein (0.001–1.0 µg) and DLPs (0.25 µg per well) were added. The amount of DLPs bound was detected by ELISA (Jagannath et al., 2006).
Transmission electron microscopy (TEM).
Purified N72 was dialysed against 10 mM Tris/HCl (pH 7.2) containing 100 mM NaCl, and approximately 10 µl of the protein was adsorbed for 1 min onto a carbon-coated 200-mesh copper grid (Structure Probe). The unadsorbed solution was removed using filter paper and the grid was washed with 10 µl double-distilled water and stained for 30–45 s with 0.5 % uranyl acetate. The grid was examined with a JEOL 100 CXII microscope at 80 kV.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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N72 from strains SA11 and Hg18 was approximately 17–20-fold more efficient in diarrhoea induction in suckling mice compared with the full-length protein (Ball et al., 1996; Jagannath et al., 2006; Table 1). Furthermore, a secreted peptide of aa 112–175 has been reported to be similar to the full-length protein in diarrhoea induction (Zhang et al., 2000), indicating that the synthetic peptide lacked either the correct conformation or the regions necessary for optimal diarrhoea induction and that the N-terminal boundary of enterotoxigenic activity could lie around aa 112. With a view to determining the C-terminal boundary of the optimal diarrhoea-inducing region and the role of the flexible C terminus in diarrhoea induction, several amino acid substitution and deletion mutants of the protease-sensitive C terminus in the context of the highly active N72 from strains SA11 and Hg18 were evaluated. As shown in Table 1, the single, double and multiple amino acid mutants as well as the deletion mutants of the flexible C terminus of SA11 or Hg18 N72 exhibited approximately 20–2080-fold increases in DD50 depending on the mutation. Furthermore, the effect of the double amino acid mutations P165S/P168A, Y131S/T139S and the triple amino acid mutation P165S/P168A/M175I was very similar to that of the deletion of 94 aa from the N terminus (N94) (Table 1). N72 from both strains induced spontaneous diarrhoea within 30–45 min of administration, with a mean diarrhoeal score of 3.2 at or above a dose of 0.05 nmol. None of the other mutants used in this study induced diarrhoea spontaneously and diarrhoeal stools were observed between 1 and 3 h after administration only after gentle pressing of the abdomen, which is similar to that induced by N72 below 0.05 nmol. Except for N85, identical mutants from the two strains exhibited very similar DD50 (Table 1). For example, the Hg18 and SA11 N94C29 mutants, which correspond to the synthetic peptide aa 114–135, showed similar DD50 values of 65, 63 and 79 nmol (Ball et al., 1996), suggesting that our results are comparable to those of other studies. Hence, similar mutants from both strains were considered equivalent for discussion.
Comparative sequence analysis revealed that the prototype asymptomatic human strains contained mutations in the flexible C terminus that were unique to each strain in nature and position in comparison with many symptomatic strains (Jagannath et al., 2000; Lin & Tian, 2003). For example, whilst the asymptomatic strains I321 and 116E contained lysine at positions 157 and 160, respectively, in place of a conserved acidic residue, strain ST3 contained arginine at position 162 instead of glycine (Fig. 2). In addition, ST3 and 116E contained a serine at position 169 instead of a basic amino acid, and I321 differed at a few other positions (Fig. 2). As amino acid substitutions and deletions in the C terminus resulted in significant increases in DD50, we sought to evaluate the effect of incorporation of the asymptomatic strain-specific substitutions into the flexible C terminus of the highly diarrhoeagenic SA11 N72 on DD50 (Table 1). Whilst the I321-specific mutant E157K exhibited an approximately 20-fold increase in DD50, the ST3- and 116E-specific double amino acid mutants G162R/R169S and E160K/R169S and the multiple amino acid mutant E157K/E160K/G162R/R169S, which contained substitutions specific for all three asymptomatic strains, showed 200–800-fold increases in DD50 in comparison with SA11 N72. Furthermore, the Y166S mutation in the flexible C terminus and Y85S mutation in the N-terminal AAH73–85 resulted in approximately 200- and 100-fold increases in DD50, respectively (Table 1).
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N72 resulted in 20–400-fold increases in DD50 in comparison with N72, double or multiple amino acid substitutions in the C terminus or deletion of 94 aa from the N terminus resulted in 200–2087-fold increases depending on the strain (Table 1). Of significance, the tremendous 10 833–12 600-fold increase in DD50 of the double deletion mutant N94C29 with respect to N72 from both strains was similar to that reported for the synthetic peptide aa 114–135 (15 800-fold, Table 1) (Ball et al., 1996). These results suggest that both the helical N terminus in N72 (Jagannath et al., 2006) and the flexible C terminus are required for optimal diarrhoea-inducing activity, and suggest that mutations at different positions after aa 72 can have major effects on the diarrhoea-inducing ability of NSP4, with some exerting more severe effects than others. Comparison of DD50 of the N-terminal mutants N85 and N94 with that reported for the secreted peptide aa 112–175, which was as efficient as the full-length protein (Zhang et al., 1998), suggests that the region between aa 85 and 94, and probably aa 112, functions as a negative modulator of diarrhoea induction. Of note, Hg18 N85 was about 10-fold less effective than SA11 N85. Purified recombinant NSP5 or PBS, used as negative controls, did not induce diarrhoea.
Amino acid substitutions and deletions in the flexible C-terminal region affect DLP binding
Earlier studies have indicated that the region of 20 aa at the C terminus is sufficient for DLP binding when fused to GST (O'Brien et al., 2000). However, whether this 20 aa peptide alone without fusion to GST functions as a DLP receptor is not known, although a C-terminal peptide failed to inhibit the receptor activity of the full-length protein (Au et al., 1993). Furthermore, deletion of the N-terminal 85 aa or mutations in AAH73–85 in the context of N72 severely affected DLP binding at low concentration, although it could be significantly restored at a higher receptor concentration, suggesting a role for the N-terminal helical region in efficient DLP binding (Jagannath et al., 2006). Iodination of microsome-bound NSP4 from virus-infected cells also abolished DLP binding, suggesting the importance of some tyrosines in DLP interaction and the presence of a second site of DLP binding in NSP4 (Au et al., 1989). Recent results from our laboratory have suggested that efficient DLP binding is dependent on a unique conformation of the cytoplasmic tail (Jagannath et al., 2006). Hence, we sought to examine the influence of a large number of amino acid mutations and deletions in the flexible C terminus (Fig. 3a and b) on DLP binding. As shown in Fig. 3, P168A, Y166S and M175I substitution mutants of the primary DLP-binding site or the C-terminal deletion mutants of N72 completely lost the ability to bind DLPs. However, the Y85S, P165S and Y131S/T139S mutants, although they failed to bind DLPs at low concentration, exhibited approximately 25, 40 and 70 % of the activity, respectively, of WT N72 at the highest concentration of 1.0 µg of receptor (Fig. 3c). Of note, identical mutants of both SA11 and Hg18 exhibited similar DLP-binding properties. These results indicated that mutations in the C-terminal region affected both diarrhoea-inducing and DLP-binding properties.
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N72 from Hg18 and exhibits high fluorescence upon binding to the protein, but highly reduced or loss of binding to the mutants of the helical N terminus, ISVD or extreme C terminus (Jagannath et al., 2006). As the C-terminal deletions and amino acid substitutions resulted in altered diarrhoea-inducing and DLP-binding properties, the conformational integrity and ability of the mutants to bind ThT in comparison with WT N72 was examined. As shown in Fig. 4(a), whilst the Y85S, Y166S and Y131S/T139S mutants exhibited a 3–5-fold reduction in ThT fluorescence, the P165S and P168A mutants showed 8-10-fold reduced ThT binding, with r2 for the ThT-binding proteins ranging from 0.800 to 0.982. However, the double proline mutant P165S/P168A, the triple mutant P165S/P168A/M175I and the C-terminal deletion mutants failed to exhibit any fluorescence, suggesting that mutations in the flexible C terminus affect the ThT-recognizable conformation. Although, the ThT emission spectra of Hg18 N72 and SA11 N72 were similar, the former consistently showed higher fluorescence; in Fig. 4(a), only that of the former is shown.
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, a significant reduction in -helical content from 61–62 % in WT SA11 or Hg18 N72 to 30–57 % in different mutants with a concomitant increase in β-sheet content from about 7 to 11–23 % was observed. These and previous results (Jagannath et al., 2006) indicate that mutations in either the C or N terminus exert a similar effect on the conformation of the CT.
Recently, we showed that an N-terminal His-tagged fragment of Mr 9950 (9.95 kDa) corresponding to aa 73–146 of the ordered multimeric forms of the highly diarrhoeagenic N72 from Hg18 and SA11 remained resistant to trypsin, despite containing multiple cleavage sites. By contrast, mutants of the helical N terminus were highly susceptible to degradation, the degree of susceptibility varying among different mutants (Jagannath et al., 2006). As different mutants of the C terminus showed altered conformation and biological properties, we sought to examine the relative trypsin resistance of the mutants in comparison with the WT N72. Surprisingly, whereas the P168A mutant was completely degraded by trypsin within 5 min at 37 °C, all other mutants showed varying degrees of resistance that were intermediate to that exhibited by P168A and WT N72 (Fig. 5).
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-helical content, ThT binding and/or trypsin resistance of different mutants suggested that the majority of mutations in the flexible C terminus affected the conformation and stability of the N-terminal tetrameric CCD of the CT.
Mutations in the flexible C terminus result in altered multimerization of the CT
Previous studies have demonstrated that SA11 NSP4 exists as oligomers and high-molecular-mass complexes (Maass & Atkinson, 1990; Taylor et al., 1992) as well as proteolytically cleaved, membrane-anchored or secreted forms in infected or NSP4 gene-transfected cells (Bugarcic & Taylor, 2006; Storey et al., 2007; Zhang et al., 2000). Prior to our studies, NSP4 was primarily considered a tetramer and the significance of the high-molecular-mass complexes in infected cells has not been investigated. Our recent studies revealed that N72 from SA11 and Hg18 containing the AAH73–85 existed as highly soluble high-molecular-mass complexes within E. coli, as observed by native PAGE and Western blotting. Deletion of 85 or 94 aa from the N terminus totally abolished multimerization, and the mutants N85 and N94 formed only tetramers. Amino acid substitutions in the N-terminal AAH73–85, the ISVD, the extreme C-terminal methionine or deletion of 5 aa from the C terminus resulted in altered multimerization/unstable multimers (Jagannath et al., 2006).
TEM of the freshly purified WT Hg18 N72 revealed ordered reticulate structures (Fig. 6) but no amorphous aggregates. Similar structures were seen for SA11 N72 (data not shown). These results demonstrated that the high-molecular-mass complexes of Hg18/SA11 N72 are highly ordered multimers. Determination of the crystal structures of different non-multimerizing mutants of the complete CT or cryoelectron microscopy of the reticulate structures is required to understand the structural organization in detail. TEM failed to reveal any structures for the non-multimerizing mutants N85 and N94.
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N72 at two different concentrations (0.6 and 2.0 mg ml–1) by SEC revealed six groups among the mutants that differed in the nature/degree of multimerization (Table 2). The majority of the P168A, P165S/P168A/M175I and Y131S/T139S mutant proteins existed as tetramers (79.0–86.0 %) and about 14–24 % as multimers at both concentrations. The second group, represented by P165S, P165S/P168A and M175I mutants, showed negligible multimers at low concentration but exhibited concentration-dependent multimerization. About 70.0 % of the Y166S mutant, representing the third group, existed in multimeric form at low concentration, but showed efficient multimerization at high concentration but with altered conformation as revealed by CD spectroscopy and ThT binding. The fourth group, represented by Y85S and T139S mutants, predominantly existed in multimeric form (75–82 %) at low concentration without significant change with an increase in concentration. Whilst Hg18 N72C5, representing the fifth group, exhibited approximately equal proportions of multimeric and oligomeric forms at both concentrations, Hg18 N72C12 and Hg18 N72C29, which represented the sixth group, although existing predominantly as high-molecular-mass complexes, differed significantly in conformation and ThT binding, precipitated upon dialysis/concentration unlike all of the other mutants, were relatively unstable compared with N72 in native PAGE and were highly susceptible to trypsin digestion, suggesting unregulated multimerization or random aggregation. Hg18 N72C29 could not be analysed by SEC as the protein precipitated upon concentration. WT N72 exhibited efficient multimerization, even at low concentration (Table 2), and the multimers were distinct from the others in several properties. No significant differences were observed in the proportion of oligomeric or multimeric forms of different mutants upon storage.
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). In particular, whilst the M175I mutant exhibited concentration-dependent multimerization, the C-terminal deletion mutant N72C5 existed in approximately equimolar proportions between the multimeric and oligomeric states, i.e. was concentration independent. In contrast, further deletion of 7 aa (N72C12) or more (N72C29) from the C terminus resulted in inherent multimerization (Table 2). It is important to note that all of the mutants, irrespective of their multimerization pattern, were conformationally different from WT N72 as observed by altered -helical content (Fig. 4b), ThT binding (Fig. 4a) and susceptibility to trypsin (Fig. 5). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Dong et al., 1997; Tian et al., 1994), membrane destabilization/permeabilization (Newton et al., 1997; Tian et al., 1996), virus transcription (Silvestri et al., 2005) and inhibition of microtubule-mediated secretory pathways (Xu et al., 2000).
NSP4 appears to be structurally highly complex, and efforts in several laboratories to crystallize the complete CT have so far failed. The highly flexible and protease-sensitive nature of the C terminus (Deepa et al., 2007; Jagannath et al., 2006; O'Brien et al., 2000) and the multimerization-promoting activity of the N-terminal helical regions from aa 52 to 85 (Jagannath et al., 2006; Newton et al., 1997) appear to impede crystallization of the complete CT (Deepa et al., 2007). The longest region that could be crystallized and for which the structure has been determined corresponds to aa 95–146 from the SA11 and I321 strains, which conclusively showed that the N-terminal region from aa 95 to 137 exists as tetrameric CCD and that the region C-terminal to aa 137 is highly flexible, and no structure could be assigned (Deepa et al., 2007). Whether the electron-dense region followed by the lighter tail-like region in the beaded chains seen in TEM correspond to the CCD and the flexible regions, respectively, of the CT is not known.
One of the most intriguing observations was the high-affinity binding of ThT to the multimeric forms of the highly diarrhoeagenic SA11/Hg18 N72, but highly reduced or total loss of binding to the tetrameric forms or to any of the large number of mutants (including those that multimerized but had altered conformation) studied in the present and previous studies (Jagannath et al., 2006). The fact that ThT bound only the ordered multimers of N72, and that every mutation in its helical N terminus, the ISVD or the flexible C terminus affected the conformation, multimerization, ThT-binding, DLP-binding, diarrhoea-inducing and/or trypsin-resistance properties, suggest that a ThT-recognizable unique conformation (Jagannath et al., 2006) correlates with ordered multimerization and optimal biological functions of the purified NSP4 proteins. ThT is known to bind β-amyloid peptides and ordered polymeric structures in proteins (Devlin et al., 2002; LeVine, 1993; Naiki et al., 1989). However, studies on acetylcholinesterase (Ferrari et al., 2001) and Hg18 N72 (Jagannath et al., 2006) have indicated that ThT can bind proteins in a conformation-specific manner in the absence of β-sheet structures. Due to the possible conformational complexity, it is difficult to predict the presumptive ThT-binding domain in NSP4.
Another interesting observation was the total loss of DLP binding by the P168A, Y166S and M175I mutants in contrast to the Y85S, Y131S and P165S mutants, which exhibited severe loss of binding at low concentration, but regained 25–70 % of the activity of that of WT N72 at higher concentration, which is similar to that exhibited by the N-terminal deletion mutants and amino acid substitution mutants of AAH73–85 or the ISVD (Jagannath et al., 2006). It is noteworthy that the mutants required 100–400-fold more protein to bind the same amount of DLPs that bound to 0.001 µg of the WT N72 (Fig. 3). These results suggest that, whilst 10 aa from the C terminus including Met175, Pro168 and Tyr166 are critical determinants, Tyr131, Pro165, the ISVD and the N-terminal helical region that includes Tyr85 determine the affinity of DLP interactions at a low concentration of the receptor, but in a manner that is totally dependent on the extreme C terminus. It should be noted that, except for murine strains, prolines at positions 165 and 168 and tyrosines at positions 85 and 166 are highly conserved in NSP4 from different strains. Concentration-dependent binding of the Y85S and Y131S mutants to DLP indicates that these residues, in particular Tyr85, are involved in the cooperativity of binding between the receptor and the ligand that involves multiple binding sites on the DLP. These results are in support of the previous observation of loss of DLP binding to iodinated microsome-associated NSP4 (Au et al., 1989), but reveal a lack of an independent second site of interaction. The primary DLP interaction at the extreme C terminus appears to induce a conformational rearrangement in the CT, generating a secondary site of interaction in the N-terminal region resulting in enhanced affinity and cooperativity of binding between the receptor and the ligand. Determination of the crystal structure of the receptor–DLP complex is required to understand the complex interaction.
An unexpected observation was the effect of single amino acid substitutions in the flexible C terminus, including that of the extreme C-terminal methionine, on the conformation of the highly stable N-terminal CCD, as observed by its altered trypsin resistance and significant decrease in -helical content with concomitant increase in β-sheet content. Whilst amino acid substitutions might affect the conformation of the CT, deletions at either of the termini appear to relieve the constraint on the ordered and regulated cooperation between the two termini, resulting in inherent multimerization but with altered conformation.
NSP4 exhibits a high degree of sequence variation in the C-terminal region from aa 135 to 175 compared with other regions in the protein (Jagannath et al., 2000). Analyses of NSP4 sequences, however, have failed to reveal any correlation between amino acid substitution and the symptomatic and asymptomatic phenotype of the virus (Angel et al., 1998; Chang et al., 1999; Jagannath et al., 2000; Kirkwood et al., 1996; Lin & Tian, 2003; Oka et al., 2001; Ward et al., 1997). Furthermore, purified recombinant NSP4s from a limited number of strains exhibit a wide variation in DD50 in suckling mice (Ball et al., 1996; Horie et al., 1999; Mori et al., 2002; Zhang et al., 1998). The present study revealed that amino acid substitutions at different positions in the flexible C terminus of the highly diarrhoeagenic SA11 NSP4 resulted in significant increases in DD50, albeit to different extents. As the biological functions of NSP4 appear to depend on a unique conformation of the CT (Jagannath et al., 2006), it is likely that mutations that result in altered virulence would differ for different NSP4s and depend on the overall sequence context of the CT. Although no linear correlation between ThT fluorescence and diarrhoea-inducing efficiency was observed, efficient diarrhoea inducers consistently exhibited high ThT fluorescence, and a negligible, or lack of, ThT binding corresponded to a high DD50 of the mutants.
It is possible that some NSP4s require interaction with other viral or cellular proteins for conformational maturation in vivo. In this context, the cytoplasmic region is known to interact with viral proteins VP4 and VP6 (Estes, 2001) and cellular proteins calnexin (Mirazimi et al., 1998), tubulin (Xu et al., 2000), laminin-β3 and fibronectin (Boshuizen et al., 2004) and caveolin (Parr et al., 2006). Recently, Hsp70 has been implicated in rotaviral protein degradation (Broquet et al., 2007). The present results should serve as a valuable guide in designing strategies for the future generation of mutant viruses using reverse-genetics approaches (Boyce & Roy, 2007; Kobayashi et al., 2007; Komoto et al., 2006) to study NSP4-mediated rotavirus virulence. In conclusion, the flexible C terminus, hitherto thought to have no significant role in diarrhoea induction, is an important modulator of the biological properties of rotavirus enterotoxin, mutations in which could significantly affect the biological functions of the protein.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 25 November 2007;
accepted 25 February 2008.
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